MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are emerging as key players in the control of fundamental biological processes in both physiological and pathological conditions. Accumulating evidence suggests that miRNAs may control specific functional pathways by targeting gene networks of functionally correlated genes. In humans, deregulation of miRNA expression caused by mutations in either the miRNA itself or its target gene has been correlated with a number of pathological conditions such as diabetes, neurodegenerative diseases, heart failure and hereditary deafness (1, 2), among others. Recently, miRNAs are also emerging as new targets of therapeutic interventions for a variety of diseases. A therapeutic role of miRNAs has already been described in a number of cancer models (3, 4), in heart diseases (5, 6), in muscular dystrophy (7) and in liver disorders (8, 9). The use of a miR-122 inhibitor has already entered the clinic, where it is in phase I trials with the goal of treating hepatitis C infection (10). Therefore, the therapeutic use of miRNAs represents a promising field of research in modern medicine, although its extensive application requires an adequate understanding of the gene expression changes controlled by miRNAs.
The retina is a layered structure composed of six neuronal and one glial cell type, which are organised in three cellular layers: the ganglion cell layer, comprising retinal ganglion (RGC) and displaced amacrine cells, the inner nuclear layer (INL), which contains bipolar, horizontal and amacrine interneurons and Müller glial cells, and the outer nuclear layer (ONL), where rod and cone photoreceptors are located. The retina is immediately adjacent to the retinal pigment epithelium (RPE), a pigmented cell layer that nourishes retinal visual cells, and is firmly attached to the underlying choroid and overlying retinal visual cells (FIG. 20).
Inherited retinal dystrophies (IRDs) represent one of the most frequent causes of genetic blindness in the western world. The primary condition that underlies this group of diseases is the degeneration of photoreceptors, i.e., the cells that convert the light information into chemical and electrical signals that are then transmitted to the brain through the visual circuits. There are two types of photoreceptor cells in the human retina: rods and cones. Rods represent about 95% of photoreceptor cells in the human retina and are responsible for sensing contrast, brightness and motion, whereas fine resolution, spatial resolution and color vision are perceived by cones.
IRDs can be subdivided into different groups of diseases, namely Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), cone-rod dystrophies and cone dystrophies.
RP is the most frequent form of inherited retinal dystrophy with an approximate frequency of about 1 in 4,000 individuals (11). At its clinical onset, RP is characterized by night blindness and progressive degeneration of photoreceptors accompanied by bone spicule-like pigmentary deposits and a reduced or absent electroretinogram (ERG). RP can be either isolated or syndromic, i.e., associated with extraocular manifestations such as in Usher syndrome or in Bardet-Biedle syndrome. From a genetic point of view, RP is highly heterogeneous, with autosomal dominant, autosomal recessive and X-linked patterns of inheritance. A significant percentage of RP patients, however, are apparently sporadic. To date, around 50 causative genes/loci have been found to be responsible for non-syndromic forms of RP and over 25 for syndromic RPs (RETnet web site: http://www.sph.uth.tmc.edu/RetNet/).
LCA has a prevalence of about 2-3 in 100,000 individuals and is characterized by a severe visual impairment that starts in the first months/years of life (12). LCA has retinal, ocular as well as extraocular features, and occasionally systemic associations. LCA is inherited as an autosomal recessive trait in the large majority of patients, while autosomal dominant inheritance has been described only in a limited number of cases. LCA is genetically heterogeneous and, to date, mutations have been identified in 15 different genes: GUCY2D (locus name: LCA1), RPE65 (LCA2), SPATA7 (LCA3), AIPL1 (LCA4), LCA5 (LCA5), RPGRIP1 (LCA6), CRX (LCA7), CRB1 (LCA8), CEP290 (LCA10), IMPDH1 (LCA11), RD3 (LCA12), NMNAT1 (LCA9), LRAT (LCA14), TULP1 (LCA15), and RDH12 (LCA13). The diagnosis of LCA is established by clinical findings. Molecular genetic testing is clinically available for the 15 genes currently known to be associated with LCA. Collectively, mutations in these genes are estimated to account for approximately 40%-50% of all LCA cases, depending on the survey. Cone-rod dystrophies (CRDs) have a prevalence of 1/40,000 individuals and are characterized by retinal pigment deposits visible upon fundus examination, predominantly localized to the macular region. In contrast to typical RP, which is characterized by primary loss in rod photoreceptors, later followed by the secondary loss in cone photoreceptors, CRDs reflect the opposite sequence of events. CRD is characterized by a primary cone involvement, or, sometimes, by concomitant loss of both cones and rods that explains the predominant symptoms of CRDs: decreased visual acuity, color vision defects, photo-aversion and decreased sensitivity in the central visual field, later followed by progressive loss in peripheral vision and night blindness (13). Mutations in at least 20 different genes have been associated with CRD (RETnet web site: http://www.sph.uth.tmc.edu/RetNet/).
Cone dystrophies (CD) are conditions in which cone photoreceptors display a selective dysfunction that does not extend to rods. They are characterized by visual deficit, abnormalities of color vision, visual field loss, and a variable degree of nystagmus and photophobia. In CDs, cone function is absent or severely impaired on electroretinography (ERG) and psychophysical testing (14). Similar to the other forms of inherited retinal dystrophies, CDs are heterogeneous conditions that can be caused by mutations in at least 10 different genes (RETnet web site: http://www.sph.uth.tmc.edu/RetNet/).
As also mentioned above, IRDs are due to the degeneration and subsequent death of photoreceptor cells, primarily rods in the case of RP and LCA and primarily cones in the case of CRDs and CDs. Of interest, in RP and in most forms of LCA, rod degeneration is followed by a secondary degeneration of cones. The vast majority of genes responsible for IRDs are expressed predominantly in photoreceptors (either rods or cones). Some IRD genes are prevalently expressed in the retinal pigment epithelium. However, also in the latter case, the main consequence that derives from the dysfunction of these genes is a damage of photoreceptor function, which then translate into photoreceptor degeneration and death. For most forms of the above mentioned diseases an effective therapy is currently unavailable.
The authors are currently investigating the possible use of miRNAs as therapeutic tools in inherited retinal dystrophies. The authors have recently studied the expression pattern of miRNAs during the main stages of mammalian eye development and generated the most comprehensive up-to-date expression atlas of miRNAs in the mammalian eye (15, 16).
As a result, the authors identified a subset of miRNAs displaying significant expression levels in the mammalian eye, and among those miR-204 and miR-211. The authors started a detailed functional characterization of the latter miRNAs, using mostly in vivo models. In particular, the authors previously demonstrated, by using gain- and loss-of-function approaches in the medaka fish [Oryzias latipes (ol)] model organism, that alteration of miR-204 activity has a significant impact on multiple aspects of eye differentiation and function. In particular, morpholino-mediated ablation of miR-204 expression resulted in an eye phenotype characterized by microphthalmia and altered dorso-ventral (D-V) patterning of the retina, which causes optic coloboma (17).
Interestingly, miR-204 and miR-211 are closely related paralogs in mammals that share the same seed-region sequence and the same set of predicted targets (TargetScan) (18). They only differ by one nucleotide in mouse and two nucleotides in human.
Recently, miR-204 and miR-211 have been suggested to exert a protective effect on both the integrity of the retinal pigment epithelium (RPE) as a barrier and on preventing its abnormal proliferation (23).
On that basis, it has been proposed that the delivery of these two miRNAs to the RPE may exert a beneficial role in ocular diseases caused by abnormalities in the differentiation and proliferation of the RPE, including vitreal retinopathy, macular degeneration and diabetic retinopathy.
The application WO2010027838 refers to methods of preventing or treating detrimental retinal epithelial cell proliferation, loss of retinal epithelial cell differentiation, age-related macular degeneration and/or proliferative vitreal retinopathy by administering miR-204, miR-211, or a mixture of miR-204 and miR-211.
The document WO2009137807 concerns methods and compositions for diagnosing and/or treating, among others, vascular diseases of the eye, specifically ocular or retinal/choroidal neovascular diseases, by administering an inhibitor of miR-211. The methods involve measuring the levels of one or multiple miRNAs in patient samples and using the test results to diagnose and/or predict an optimal treatment regimen for the patient. Use of miR-211 is claimed for increasing vascularization.
Then, there is still the need for therapy that has a protective effect on the process of photoreceptor degeneration and death which are the primary conditions that underlie inherited retinal dystrophies. In such diseases, abnormal RPE differentiation and proliferation do not play key pathogenic roles.